Past studies from our laboratory have shown that, during rat testicular development, Sertoli cells synthesize and secrete a protein (S45-S35 heterodimeric protein; synonymous names: SGP-2, clusterin, TRPM-2) which then acquires a spermatogenic stage-dependent expression pattern. The purpose of this renewal application is to continue our study of S45-S35 function in spermatogenesis. During the previous funding periods, we have shown that components of the head and tail of developing spermatids and epididymal sperm contain proteins with antigenic homology to S45-S35. We have also examined the expression of S35-S45 mRNA during testicular development and spermatogenesis. During the current funding period we have found that pachytene and spermatids also express S35-S45 mRNA. However, while in Sertoli cells the somatic-type S35-S45 encoded precursor gives rise to the disulfide linked S45-S35 heterodimer, the spermatogenic-type S35-S45 gene generates a similar precursor but its post-translational processing yields monomers. We have also shown that proteins associated with both outer dense fiber (ODF) polymers of sperm tail and sperm protamine-DNA chromatin are antigenically homologous to the S45-S35 precursor. As a result of these studies we have identified structural components in sperm with a role in the morphogenesis of ODF polymers and packaging of sperm chromatin in an orderly fashion. Therefore, we are in a unique situation to analyze molecular aspects of spermiogenesis, in particular the marking genes which may be required during spermiogenesis and, eventually, at fertilization and during early embryogenesis. During the next funding period, we will address specific aspects of the structure of the spermatogenic-type S35-S45 gene and its function in sperm formation through five specific aims: (1) to determine the structure and functional regulation of the spermatogenic-type S35-S45 gene, in particular, the characterization of alternative polyadenylation and splicing sites and identification of nuclear regulatory elements controlling the in vitro transcription of somatic- and spermatogenic-type S35-S45 genes. (2) To characterize molecular aspects of the coiled-coil subunits of ODF polymers and ODF-associated protein(s) and to examine more rigorously the molecular basis for the antigenic homology of ODF-associated proteins with the common somatic and spermatogenic precursor. (3) To elucidate early stages of the assembly of the ODF polymer by in vitro reconstitution and chemical crosslinking of ODF subunits and ODF-associated protein(s). (4) Isolate sperm nuclear matrix proteins bound to a distinct portion of the sperm DNA loop, define DNA molecular characteristics and determine the primary structure of a 38.5 kD nuclear matrix protein antigenically homologous to the somatic/spermatogenic common precursor. (5) Isolate and identify DNA sequences specifically associated with segments of sperm DNA loops bound to or free of nuclear matrix proteins and approach the question of gene marking during spermiogenesis. Results from these studies are significant for understanding molecular mechanisms whereby anomalous transcripts can lead to the formation of abnormal and unfertile sperm as well as testicular malignancies.